Profective Effects And Mechanism Of Neuritin Overexpression On Repaiirng Injured Rat Optic Nerve And RGC5

Purpose:Optic nerve injury treatment is the difficult among many eye diseases and injuries.Retinal ganglion cells (RGCs) suffers a great loss and the failure of axon regenerationfollowing optic nerve crush, which then contribute to blindness. How to reverse thedilemma effectively is always one of the challenges faced by the ophthalmology andneuroscience field. Neuritin, also called candidate plasticity-related gene15(CPG15), is anovel member of the neurotrophic factors (NTs) family, which mainly expressed in thenervous system. Neuritin is a common downstream factor of neurotrophic factors andnervous activities. It plays an important role in protecting cortical neurons from apoptosis,promoting neurite outgrowth, increasing synapse maturation and modulating synapticplasticity.Based on the Neuritin’s neurotrophic and protective effect on neurotrauma anddisease, our present study investigated the spatiotemporal expression of Neuritin in retinaand optic nerve after optic nerve injury. And then Neuritin was used as a target for genetherapy which may have effect on protecting RGCs survival and promoting axonsregeneration after optic nerve injury. The investigation may contribute to reveal theunderlying mechanism of the difficulty of optic nerve regeneration and provide a newmethod to repair the injured optic nerve.Methods:(1) Animal model of the optic nerve crush: A Yasargil aneurysm clip was applied tocrush the optic nerve for9seconds approximately1mm behind the eyeball to make anincomplete injury model similar to the clinic. HE staining, CTB anterograde tracing andtransmission electron microscopy were used to observe the optic nerve’s morphologychange after injury.(2) Detection of the spatiotemporal expression of Neuritin in retina and optic nerveafter optic nerve injury: The double immunostaining was used to determine the histologicallocalization of Neuritin. Western blot and Real time PCR were used to detect thespatiotemporal expression of Neuritin mRNA and protein in retina and optic nerve afteroptic nerve injury.(3) Neuritin lentivirus was constructed and its titer was determined.(4) In vivo study of the effect of Neuritin overexpression on injured rat RGCs:Intravitreal injection of Neuritin lentivirus was processed1week before the optic nerve crush. RGCs were labeled with an injection of CTB2weeks after the optic nerve crush forobserving the survival and axonal regeneration of RGCs.(5) In vitro study of the effect and mechanism of Neuritin on protecting RGC5cellline: CCK8or MTT kit were used to detect the protective effect of Neuritin to the injuredRGC5. Phase contrast microscope was used to observe the neurite growth of RGC5, andDAPI staining and Western blot were used to detect cell apoptosis. Further more, Westernblot was used to detect the expression of neurotrophic factors related signal pathwayproteins, trying to illustrate the underlying mechanism of Neuritin on injured RGC5.Results:(1) Established the incomplete optic nerve injury model successfully: The HE stainingresults showed that the epineurium in injured area was intact after optic nerve crush.Appearance of CTB-positive axons in the distal region of the injured nerve suggested somenormal axons were maintained and had transporting function. Some axons in the opticnerve before the optic chiasma still appeared normal like morphology observed under thetransmission electron microscope.(2) The histological localization of Neuritin in retina and optic nerve: Doubleimmunostaining results showed that Neuritin mainly expressed in RGCs in normal retinaincluding its cell body and neurite and part of the astrocytes. In normal optic nerve,Neuritin was mainly observed in RGCs axons, oligodendrocytes and vascular endothelialcells.(3) The spatiotemporal expression of Neuritin in retina and optic nerve after opticnerve injury: Western blot results showed that Neuritin protein expression transientlyupregulated at1d and reached its peak at1d, remained high levels at3d and7d, thendeclined to a basal level in both retina and optic nerve. Similarly, real time PCR resultsshowed that the level of Neuritin transcripts transiently reached its peak expression at1d,and downregulated dramatically to a even lower basal level.(4) Neuritin lentivirus plasmid was constructed and and a high titer of Neuritinlentivirus was obtained successfully.(5) In vivo study of the effect of Neuritin overexpression on injured rat RGCs:Neuritin overexpression in vivo in RGCs increased the number of survival RGCs and theirregenerated axons after optic nerve crush for2weeks.(6) In vitro study of the effect and mechanism of Neuritin on protecting RGC5cell line:CCK8detection showed that recombinant human Neuritin protein(rhNeuritin) increased the livability of damaged RGC5. Overexpression of Neuritin increased the longest neuritelength of RGC5observed by phase contrast microscope and decreased the apoptosis ratesand the expression of cleaved Caspase3expression under the detection of DAPI stainingand Western blot. In addition, the present study revealed first time that Neuritin couldinhibit the expression of pSTAT3protein and up-regulated the expression of pAKT andpERK proteins in the injured RGC5.Conclusions:(1) A Yasargil aneurysm clip can be successfully used to establish the incompleteoptic nerve injury model.(2)Neuritin was mainly expressed in RGCs and partial astrocytes in normal retina andin RGCs axons, oligodendrocytes and vascular endothelial cells of normal optic nerve.(3) Transiently early increasing and then subsequently declining and deficiency ofNeuritin expression in retina and optic nerve after crush may be one of the critical reasonswhich contribute to the RGCs death and the failure of axonal regeneration.(4) In vivo, we first confirmed that Neuritin overexpression can promote the survivalof RGCs and stimulate its axonal regeneration effectively after optic nerve crush.(5) In vitro, we further confirmed that rhNeuritin or Neuritin lentivirus can protectedthe injured RGC5effectively, by which can raise the survival rate, reduce the cell apoptosis,and promote the neurite growth of injured RGC5cells. This protective effect is mayaccomplished through inhibiting STAT3pathway and upregulating AKT and ERKpathways.